Role of rhodopsin phosphorylation at multiple sites <i>in vivo</i>

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  • Ohguro Hiroshi
    Department of Ophthalmology. Hirosaki University School of Medicine
  • Ohguro Ikuyo
    Department of Ophthalmology. Hirosaki University School of Medicine
  • Nakazawa Mitsuru
    Department of Ophthalmology. Hirosaki University School of Medicine

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Other Title
  • 複数のリン酸化部位を有するロドプシンリン酸化の生体内における役割
  • Role of Rhodopsin Phosphorylation at Multiple Sites in Vivo

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Abstract

    To study rhodopsin (Rho) phosphorylation at multiple sites in vivo, antibodies toward major Rho phosphorylation sites in vivo, 334Ser, 338Ser and 343Ser were prepared by immunization of authentic phosphorylated peptides to rabbit. Purified antibodies specifically recognized either the phosphorylated-334Ser, -338Ser or 343Ser site by ELISA. Immunofluorescence labeling by all these antibodies was exclusively light-dependent. However, their labeling patterns during light and dark adaptation were different from each other. Anti-P-Rho334 and P-Rho338 antibodies reacted with retinal photoreceptor outer segments (ROS) adapted by 650 and 5000 lux which caused Rho bleach at approximately 7 and 30% levels, respectively. However. in contrast, positive immunofluorescence labeling of ROS by anti-P-Rho343 was observed only in 5000 lux light-adapted rat retina. Similarly, strong, dim and negative stainings of ROS by anti-P-Rho334, P-Rho338, and P-Rho343 antibodies, respectively, were recognized in light adapted human retina by surgical illumination (approximately at 2000 lux) obtained from a patient with orbital malignant tumor. During dark adaptation of rat retina from light adaptation, immunoreactivities toward anti-P-Rho343 had diminished the fastest, followed by anti-P-Rho338 and then anti-P-Rho334. Our present results indicated that this newly developed method using specific antibodies toward different sites of phosphorylation is suitable for analysis of Rho phosphorylation and dephosphorylation in vivo.

Journal

  • Hirosaki Medical Journal

    Hirosaki Medical Journal 56 (2-4), 61-68, 2005

    Hirosaki University Graduate School of Medicine,Hirosaki Medical Society

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